Compositions and methods for cancer detection and treatment

ABSTRACT

Disclosed herein are compositions and methods for cancer detection and treatment. Compounds that inhibit PRMT5 are contemplated, as are pharmaceutical compositions comprising a therapeutically effective amount of at least one PRMT5 inhibitor. In some embodiments pharmaceutical compositions further comprising at least one HDAC inhibitor are contemplated. Methods of treating disorders in a mammal by inhibiting PRMT5 by administering to a mammal, a therapeutically effective amount of a PRMT5 inhibitor are also disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/289,301, filed on Dec. 22, 2009, and U.S. Provisional Application No.61/293,890, filed on Jan. 11, 2010, which are both incorporated byreference herein in their entirety.

BACKGROUND

High grade astrocytomas are the most common primary central nervoussystem (CNS) malignancy afflicting nearly 10,000 pediatric and adultpatients each year in North America alone. While surgery remains themost effective curative therapy for CNS tumors, grade III (anaplasticastrocytoma) and grade IV (glioblastoma, GBM) astrocytomas exhibit ahighly invasive histology that precludes effective surgical resection.Consequently, GBM tumors are among the most malignant cancers with amean survival of approximately one year despite multimodal therapy withsurgery, radiation and chemotherapy. In contrast to many cancers, thesurvival outcome of patients diagnosed with GBM has improved onlymarginally over the past several decades. While our understanding of thepathophysiology of these high grade malignancies has improved over thepast several years, discovery of effective therapies have been limitedas few novel targets affecting the complex pathways dysregulated in GBMhave been identified.

Malignant astrocytomas display an impressive spectrum of genetic andepigenetic abnormalities affecting multiple pathways relevant to cellgrowth, survival and remodeling of the tumor microenvironment. Over thepast several years, numerous genome-wide studies have demonstrated thatGBM possesses a remarkable degree of heterogeneity with regard togenetic mutation, gene expression profiles, and epigeneticmodifications. This degree of biologic heterogeneity has undoubtedlycontributed to the challenge of identifying key proteins that arecritical to the underlying pathogenesis of this aggressive disease.

Posttranslational modification of proteins is a common activity involvedat virtually all levels of cellular regulation. The enzymes thatcovalently modify amino acids add an additional layer of regulatorycontrol over multiple cellular processes including chromatin remodeling,gene transcription, signal transduction, DNA repair and RNA processing.The enzymes of the protein arginine methyltransferase (PRMT) familyrepresents a group of proteins that are evolutionarily conserved amongsta wide variety of organisms. PRMT enzymes covalently modify both histoneand a growing number of proteins that are critical to the maintenance ofnumerous cellular regulatory networks. The PRMT5 enzyme is a type IIarginine methyltransferase that utilizes the donor moleculeS-adenosyl-L-methionine to catalyze the transfer of a methyl group totwo of three guanidino nitrogen atoms within the arginine molecule.PRMT5 drives the formation of both ω-monomethylarginine and symmetricdimethylarginine residues to affect a wide range of key biologicfunctions at the level of chromatin to control transcriptionalrepression and as an enzyme that modulates non-histone protein function.

In addition to classic gene mutations, epigenetic silencing of tumorsuppressor genes (TSG) frequently leads to dysregulation of signalingpathways and promotion of tumorigenesis. Chromatin remodeling enzymeslike histone deacetylase (HDAC), DNA methyltransferase, and proteinarginine methyltransferase 5 (PRMT5) are involved in silencing TSGexpression and over expression of these enzymes can promote cellulartransformation. Drugs that inhibit HDAC enzymes (HDAC-I) or prevent DNAmethylation are currently being examined in clinical trials treatingpatients with cancer. HDAC-I and hypomethylating agents had been shownto possess anti-tumor activity in malignant gliomas both in vitro and invivo, however, clinical trials investigating these agents have beendisappointing, pointing to the need to identify other promisingepigenetic targets. PRMT5 has been shown to methylate histone proteinsat H3 arginine 8 (H3R8) and H4R3, and trigger TSG silencing. PRMT5 haseither a positive or negative effect on its substrates by argininemethylation when interacting with a number of complexes and is involvedin a variety of cellular processes, including RNA processing, signaltransduction, transcriptional regulation, and germ cell development.Recent studies showed PRMT5 to be a major pro-survival factor regulatingeIF4E expression and p53 translation. PRMT5 depletion triggersp53-dependent apoptosis and sensitized various cancer cells to Tumornecrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) withoutaffecting TRAIL resistance in non-transformed cells.

Several groups have recently described how PRMT5 dysregulation canaffect cell growth. Work previously reported demonstrates thatoverexpression of PRMT5 is involved in the pathogenesis of mantle celllymphoma, an aggressive hematologic malignancy. While that workcontributed information regarding the mechanism of PRMT5 overexpression,the functional consequences of PRMT5 inhibition on growth and survivalof the transformed cell remain poorly characterized. We have found thatthe PRMT5 enzyme is overexpressed in cell tines derived from high gradeastrocytomas and in primary anaplastic astrocytoma and GBM tumors. Thedegree of PRMT5 overexpression was related to proliferative index ofboth cell lines and primary tumors and was found to be an independentprognostic factor that could identify patients with more aggressivedisease and poor overall survival. Because this enzyme is intimatelyinvolved with numerous processes that are frequently dysregulated incancer, we were led to evaluate the consequences of PRMT5 inhibition inhigh grade astrocytomas as these cancers display overexpression of PRMT5in the context of profound molecular heterogeneity. We found thatinhibition of PRMT5 overexpression in GBM cells led to restoration ofcritical regulatory pathways affecting cell growth, survival, and tumorsuppressor and immune modulatory gene expression. These findingssuggested that experimental therapeutic strategies aimed at inhibitingthe effects of PRMT5 overexpression in cancer might lead to a betterunderstanding how to directly and indirectly affect GBM tumorprogression.

Identification of novel therapeutic strategies to improve the outcome ofpatients with high grade astrocytomas has, for the most part, provedelusive to date. This is due to several problems inherent to high gradegliomas including: (1) the degree of molecular heterogeneity; (2) lackof a universal target selectively expressed in the tumor; (3) invasivenature of the disease; (4) rapid evolution of chemo and radiationresistance; and (5) likelihood that high grade gliomas arise as aconsequence of cancer stem cell and clonal evolution. Identification ofa central factor driving these multiple pathways contributing to growth,survival, invasiveness and resistance has proven difficult.

PRMT5 overexpression in high grade gliomas can serve as an attractivetherapeutic target for several reasons. First, PRMT5 is selectivelyoverexpressed in high grade gliomas and not in normal human astrocytesor neighboring primary brain tissue. Second, PRMT5 knock down results incell cycle arrest, apoptosis, reduced invasiveness and restoration oftumor suppressor and immune modulatory cytokine gene expression. Third,PRMT5 is over expressed in glioma brain tumor stem cells. PRMT5overexpression is highly relevant to high grade glioma biology andtherefore fulfills the criteria of an optimal target for this and otherdiseases.

DESCRIPTION

High grade astrocytomas are aggressive brain to ors that are associatedwith a dismal prognosis and are considered incurable with a meansurvival of less than one year despite intensive multimodality therapy.The limits of current treatment modalities indicate a need forinnovative therapies targeting grade III and grade IV (glioblastomamultiforme, GBM). Recent studies have shown that post translationalcovalent modification of proteins and epigenetic regulation of chromatinplays a central role in the control of cell growth, differentiation, andproliferation. Chromatin remodeling enzymes like histone deacetylase(HDAC), DNA methyltransferase and protein arginine methyltransferase 5(PRMT5) are involved in silencing pro inflammatory and tumor suppressorgene (TSG) expression and contribute towards cellular transformation.The PRMT5 enzyme contributes towards transcriptional silencing ofseveral important regulatory genes by methylating arginine residues onhistone proteins (at histone 4 arginine residue 3 (H4R3) and (H3R8)).

It is disclosed herein that epigenetic processes driven by PRMT5 overexpression are relevant in regulation of key oncogenic pathways that areoperable in high grade astrocytomas. Patient-derived GBM cell lines andprimary GBM tumors over express abundant levels of PRMT5 protein. Normalbrain tissue and low or intermediate grade astrocytomas do not overexpress PRMT5 suggesting that overexpression may selectively occur inhigh grade, more aggressive gliomas. The degree of PRMT5 over expressioninversely correlated with survival of GBM patients and withproliferation of GBM cell lines.

Small inhibitory RNA molecules (siRNA) are disclosed herein to inhibitPRMT5 expression leading to demethylation of target histone proteinarginine residues and transcriptional de-repression and translation oftumor suppressor and immune modulatory gene products Inhibition of PRMT5overexpression led GBM cells to undergo cell cycle arrest, apoptosis,and complete inhibition of cell migration. PRMT5 knockdown led GBM celllines to become sensitized to the toxic effects of temazolomide, a drugconsidered standard of care in upfront therapeutic strategies to treatpatients with GBM. It is believed that shown herein is that PRMT5 isboth an important prognostic factor and an attractive therapeutic targetfor GBM. Computational modeling systems utilizing crystallographicstructure of homologous PRMT enzymes allowing for construction of amolecular model of PRMT5 and rapid screening of over 10,000 smallmolecule compounds have been developed. This method has led to thediscovery of small molecules that inhibit PRMT5 activity. These novelstrategies can be used to generate more potent and selective smallmolecule inhibitors of PRMT5 activity. Promising compounds can berigorously evaluated on both in vitro and in vivo development platformsand will enhance the ability to discover a new class of drugs thatselectively inhibit a promising therapeutic target in GBM.

It is believed that PRMT5 overexpression is an oncogenic processassociated with more aggressive clinical behavior and poor overallsurvival of patients with high grade astrocytomas. Using siRNA moleculesto block PRMT5 expression supports the experimental therapeutic approachto inhibit PRMT5 in GBM.

Additional features and advantages will be set forth in part in thedescription that follows, and in part will be obvious from thedescription, or may be learned by practice of the inventions. Theobjects and advantages of the inventions will be realized and attainedby means of the elements and combinations particularly pointed out inthe appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the inventions, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate some embodiments of theinventions, and together with the description, serve to explainprinciples of the inventions.

FIG. 1 shows epigenetic repression of anticancer genes and rationale fortargeting PRMT5. (A) Both activating and repressive SWI/SNF complexesco-exist in the cellular proteome. Hypoacetylation of histones H3 and H4promotes PRMT5-driven methylation of arginine residues resulting incondensed nucleosomal structure and repression of target tumorsuppressor genes. (B) Following treatment with PRMT5 inhibitors, enzymespromoting gene expression are able to access chromatin and (C) restoreexpression of key regulatory genes.

FIG. 2 shows PRMT5 is overexpressed in GBM and is inversely correlatedwith survival (A) Kaplan Meier plots of overall survival by gliomagrade. As expected, glioma grade is significantly associated withoverall survival in the cohort. (B) Distribution of PRMT5 expressionlevel by glioma grade. PRMT5 levels are significantly associated withglioma grade (Kruskal-Wallis equality-of-populations rank testp=0.0001). (C) Kaplan Meier plots of overall survival by PRMT leve inGBM. In patients with GBM there was a statistically significantassociation of PRMT5 level and overall survival (Log rank p<0.0001). (D)Time to death and PRMT5 level in patients who died with GBM. PRMT5 levelis continuously associated with time to death (Spearman's rho=−0.57,p=0.0001).

FIG. 3 shows (A) Inhibition of PRMT5 with siRNA (48 hrs) overexpressionin GBM cell lines (U1242 p53 mut, A172 p53 wt) results in apoptosis asmeasured by annexin V PI staining and flow cytometry. (B) inhibition ofmigration at 12 hrs after addition of siRNA to knock down PRMT5 vs.Scramble siRNA control.

FIG. 4 shows quantitative/qualitative PCR results using primers thatspecifically amplify promoter regions of ST7 and Chemokines CCL5, CXCL10and CXCL11.

FIG. 5 shows siRNA knockdown of PRMT5 leads to increased ST7 andChemokine transcription as measured by real time quantitative PCR (lefttop panels) and qualitative PCR (right top panel). Western blot analysisshows that this increased transcription results in enhance proteinexpression of the ST7 tumor suppressor and immune modulatory chemokinesCCL5, CXCL10 and CXCL11 (left and right bottom panels, respectively).

FIG. 6 shows SAH and arginine residue conformations docked to HumanPRMT5 model (b) as compared to crystal structure rat PRMT1 (PDB ID:1OR8) with co-crystallized SAH and substrate arginine (a). For clarity,protein molecular surface is generated omitting the residues coveringthe catalytic site face. Surface transparency is applied to showcatalytic residues. Conserved catalytic residue interactions arereproduced in docking to hPRMT5 model as displayed. Glu392 makesbifurcated hydrogen bonding with ribose hydroxyl groups whereas thesubstrate arginine residue hydrogen bonds with Glu435.

FIG. 7 shows (A) Virtual screen hits selected for validation inbio-assays. (B) Eight compounds selected for lowest binding energy froma high throughput library screen of 10,000 small molecules.

FIG. 8 shows a binding pocket where compound 5 (CMPZ5) (from FIG. 7)fits into the active site and blocks interaction between SAH and thearginine binding pocket.

FIG. 9 shows (A) Comparison of symmetric 2Me arginine methylation onH4R3 residues following a 8 hour incubation of the glioma cell lineU1242 with compounds 3 and 5. Confocal microscopy was used with mAbsspecific for symmetrical dimethylation (S2Me) of histone H4R3. Positivecontrol for loss of PRMT5 activity was provided by treatment of U1242cells with a PRMT5-specific siRNA used in above experiments. (B) Cellproliferation was assessed in replicate cultures incubated in DMSO andcompounds 1-8. Compounds 3 and 5 were the only compounds that resultedin reduced cell proliferation and H4R3 S2Me. All other compounds showedproliferation rates comparable to DMSO control conditions.

FIG. 10 shows absolute cell numbers determined at 48 hours afteraddition of either control or various concentrations of compound 5identified in the initial screen. Top figure is cell growth compared tocontrol and bottom panel shows S2Me-H4R3 content in each condition.

FIG. 11 shows that compound 5 was capable of selectively inhibitingPRMT5 activity (and not PRMT1 activity). This demonstrates that theinhibitors demonstrate selective type II PRMT inhibition.

FIG. 12 shows U1242 GBM cells were treated with (A) DMSO control, (B)HDAC inhibitor TSA (75 uM), (C) CMP5 (10 uM), or (D) combinationTSA+CMP5. Cells were stained with a mAb specific for symmetricaldimethyl arginine H4R3 and evaluated by flow cytometry (top panels) andconfocal microscopy (bottom). This figure demonstrates loss of S2Me H4R3with low concentrations of CMP5 and HDAC inhibitor TSA consistent withbiochemical synergy allowing CMP5 to inhibit PRMT5 more efficiently atlow concentrations when in the presence of HDACi.

FIG. 13 shows Compound 5 (ChemBridge ID 9033823; pink carbon, stickrepresentation) docked to the hPRMT5 model (binding energy: −8.10kcal/mol) over laid with docked SAH (Green carbon, line representation)and. The tricyclic ring system (R1) fits to the pocket making favorablevan der Waal's interaction and cation-pi interaction with the conservedamino acid residue Lys333. The substrate Arginine binding pocket (R2) isoccupied by the pyridine ring.

FIG. 14 shows 7 potential compounds with improved binding energies havebeen determined and are identified for chemical synthesis.

FIG. 15 shows virtual compounds docked to the catalytic site to evaluatethe binding energy.

FIG. 16 shows that CMP5 was capable of selectively inhibiting PRMT5activity and not PRMT1 enzymatic activity.

FIG. 17 shows the design of the analogs is based on replacing thepyridine ring of CMP5 with different substituted benzene. BLL-2-BLL-8were synthesized through a one step reductive amination reaction.

FIG. 18 shows use of single agent CMP5 (green) or TSA (blue) showed nochange in S2Me-H4R3, however combination treatment (red) showedsignificant loss of methylation.

FIG. 19 shows anti tumor, proapoptotic activity was enhanced in asynergistic fashion when low dose CMP5 was used in combination with theHDAC inhibitor TSA.

FIG. 20 shows (A) 5-aza, TSA, and CMP5 led to synergistic effects ininducing cell death. Jeko and Mino cells were treated with sub-toxicdose of 5-aza, TSA, CMP5 and combination of these drugs. Viability wasevaluated by flow cytometry. (B, C) Jeko cells were treated by 25 uM ofCMP5, 50 uM of TSA, and combo, expression of S2Me-H4R3 were detected bywestern blot (B) and confocal microscope (C).

FIG. 21 shows (left) in vitro methylation assay showed BLL36 decreasedmethyltransferase activity of PRMT5 at a lower concentration than CMP5(25 uM v.s. 100 uM). SAM was omitted for the substrate control, andhuman pure PRMT5 enzyme was omitted for blank control. (right) Jeko andMino cells were treated by 10 uM of BLL36 for 24 hr, viability wasevaluated by flow cytometry.

FIG. 22 shows optimization of CMP5. Left, R3 will be attached to CMP5 tomimic the interaction between co-factor adenine ring and PRMT5. Right,structure of CMP5 and the position where R3 will be attached.

FIG. 23 shows a novel chemical synthesis for compound 5.

DETAILED DESCRIPTION

The present inventions will now be described by reference to some moredetailed embodiments, with occasional reference to the accompanyingdrawings. These inventions may, however, be embodied in different formsand should not be construed as limited to the embodiments set forthherein. Rather, these embodiments are provided so that this disclosurewill be thorough and complete, and will fully convey the scope of theinventions to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these inventions belong. The terminology used in thedescription of the inventions herein is for describing particularembodiments only and is not intended to be limiting of the inventions.As used in the description of the inventions and the appended claims,the singular forms “a,” “an,” and “the” are intended to include theplural forms as well, unless the context clearly indicates otherwise.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the following specification andattached claims are approximations that may vary depending upon thedesired properties sought to be obtained by the present inventions. Atthe very least, and not as an attempt to limit the application of thedoctrine of equivalents to the scope of the claims, each numericalparameter should be construed in light of the number of significantdigits and ordinary rounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the inventions are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Every numerical range given throughoutthis specification will include every narrower numerical range thatfalls within such broader numerical range, as if such narrower numericalranges were all expressly written herein.

The expression “effective amount,” when used to describe an amount ofcompound applied in a method, refers to the amount of a compound thatachieves the desired pharmacological effect or other effect, for examplean amount that inhibits the abnormal growth or proliferation, or inducesapoptosis of cancer cells, resulting in a useful effect. The terms“treating” and “treatment” mean causing a therapeutically beneficialeffect, such as ameliorating existing symptoms, preventing additionalsymptoms, ameliorating or preventing the underlying metabolic causes ofsymptoms, postponing or preventing the further development of a disorderand/or reducing the severity of symptoms that will or are expected todevelop.

Contemplated herein are compounds of formula I:

wherein

-   -   R₁ is

-   -   R₂ is

-   -   A₁, A₂, A₃, A₄, and A₅ are each individually hydrogen, halo,        alkyl, alkoxyl, acetoxyl, alkylacetoxyl, —OH, trihalomethyl,        —NH₂ or —NO₂;    -   A₆ and A₇ are each individually hydrogen, OH or NH₂;    -   A₈, A₉, A₁₀, A₁₁, A₁₂, A₁₃ and A₁₄ are each individually        hydrogen, halo, alkyl, alkoxyl, acetoxyl, alkylacetoxyl, —OH,        trihalomethyl, —NH₂ or —NO₂; and    -   A₁₅ is alkyl (1-6 carbons in length); or        a salt thereof. In some embodiments the compound of formula I        may be

Also contemplated herein are compounds of formula II

wherein

-   -   R₁ is

-   -   R₂ is

-   -   R₃ is

-   -   A₁, A₂, A₃, A₄, and A₅ are each individually hydrogen, halo,        alkyl, alkoxyl, acetoxyl, alkylacetoxyl, —OH, trihalomethyl,        —NH₂ or —NO₂;    -   A₆ and A₇ are each individually hydrogen, OH or NH₂;    -   A₈, A₉, A₁₀, A₁₁, A₁₂, A₁₃ and A₁₄ are each individually        hydrogen, halo, alkyl, alkoxyl, acetoxyl, alkylacetoxyl, —OH,        trihalomethyl, —NH₂ or —NO₂; and    -   A₁₅ is alkyl (1-6 carbons in length); or        a salt thereof. In some embodiments the compound may be

Also contemplated herein are pharmaceutical compositions comprising atherapeutically effective amount of the compound of formula (I) orcompound of formula (II), in combination with a pharmaceuticallysuitable carrier.

The compounds according to formula I or II, any of the embodimentsthereof, as well as intermediates used in making compounds according toformula I or II may take the form of salts. The term “salts” embracesaddition salts of free acids or free bases which are compounds describedherein. The term “pharmaceutically-acceptable salt” refers to saltswhich possess toxicity profiles within a range that affords utility inpharmaceutical applications. Pharmaceutically unacceptable salts maynonetheless possess properties such as high crystallinity, which mayrender them useful, for example in processes of synthesis, purificationor formulation of compounds described herein. In general the usefulproperties of the compounds described herein do not depend critically onwhether the compound is or is not in a salt form, so unless clearlyindicated otherwise (such as specifying that the compound should be in“free base” or “free acid” form), reference in the specification tocompounds of formula I or II should be understood as encompassing saltsof the compound, whether or not this is explicitly stated.

Suitable pharmaceutically-acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic,.beta.-hydroxybutyric, salicylic, galactaric and galacturonic acid.Examples of pharmaceutically unacceptable acid addition salts include,for example, perchlorates and tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts include, forexample, metallic salts including alkali metal, alkaline earth metal andtransition metal salts such as, for example, calcium, magnesium,potassium, sodium and zinc salts. Pharmaceutically acceptable baseaddition salts also include organic salts made from basic amines suchas, for example, N,N-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocaine. Examples of pharmaceutically unacceptable base addition saltsinclude lithium salts and cyanate salts.

All of these salts may be prepared by conventional means from thecorresponding compound according to formula I or II by reacting, forexample, the appropriate acid or base with the compound according toformula I or II. Preferably the salts are in crystalline form, andpreferably prepared by crystallization of the salt from a suitablesolvent. The person skilled in the art will know how to prepare andselect suitable salts for example, as described in Handbook ofPharmaceutical Salts Properties, Selection, and Use By P. H. Stahl andC. G. Wermuth (Wiley-VCH 2002).

The compounds according to formula I or II, and salts thereof as well asintermediates used in making compounds according to formula I or IL andsalts thereof may take the form of solvates, including hydrates. Ingeneral, the useful properties of the compounds described herein are notbelieved to depend critically on whether the compound or salt thereof isor is not in the form of a solvate.

The compounds according to formula I or II, and salts thereof as well asintermediates used in making compounds according to formula I or II, andsalts thereof, may be administered in the form of prodrugs. By “prodrug”is meant for example any compound (whether itself active or inactive)that is converted chemically in vivo into a biologically active compoundof the formula I or II following administration of the prodrug to apatient.

Generally a “prodrug” is a covalently bonded carrier which releases theactive parent drug when administered to a mammalian subject. Prodrugscan be prepared by modifying functional groups present in the compoundsin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compounds. Prodrugs includecompounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups arebonded to any group that, when administered to a mammalian subject,cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl grouprespectively. Examples of prodrugs include, but are not limited to,acetate, formate and benzoate derivatives of alcohol and aminefunctional groups in the compounds according to formula I or II.Specifically, conjugates such as β-glucuronides and fl-galactosides havebeen suggested as prodrugs of hydroxamates. See Thomas, et al., Bioorg.Med. Chem. Lett., 2007, 983-986.

The suitability and techniques involved in making and using prodrugs arewell known by those skilled in the art. Preparation and use of prodrugsis discussed in T. Higuchi and V. Stella, “Pro-drugs as Novel DeliverySystems,” Vol. 14 of the ACS Symposium Series, and in BioreversibleCarriers in Drug Design, ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987, both of which are herebyincorporated by reference in their entirety.

The compounds of formula I or II may be administered in the form of apharmaceutical composition, in combination with a pharmaceuticallysuitable carrier. The active ingredient in such formulations maycomprise from 0.1 to 99.99 weight percent. “Pharmaceutically suitablecarrier” means any carrier, diluent or excipient which is compatiblewith the other ingredients of the formulation and not deleterious to therecipient.

The active agent may be administered with a pharmaceutically suitablecarrier selected on the basis of the selected route of administrationand standard pharmaceutical practice. The active agent may be formulatedinto dosage forms according to standard practices in the field ofpharmaceutical preparations. See Alphonso Gennaro, ed., Remington: TheScience and Practice of Pharmacy, 20th Edition (2003), Mack PublishingCo., Easton, Pa. Suitable dosage forms may comprise, for example,tablets, capsules, solutions, parenteral solutions, troches,suppositories, or suspensions.

The specific dose of a compound according to formula I or II required toobtain therapeutic benefit in the methods of treatment described hereinwill, of course, be determined by the particular circumstances of theindividual patient including the size, weight, age and sex of thepatient, the nature and stage of the disease being treated, theaggressiveness of the disease disorder, and the route of administrationof the compound.

Compounds according to formula I or II are therapeutically useful. Thereare therefore provided uses of the compounds according to formula I orII in therapy and diagnostics, and therapeutic and diagnostic methodscomprising administering a compound according to formula I or II, or apharmaceutically acceptable salt thereof, to an individual.

Also contemplated herein are compositions comprising compounds offormula I or II and HDAC inhibitors.

The compounds according to formula I or II are believed effectiveagainst a broad range of cancers and tumor types, including but notlimited to bladder cancer, brain cancer, breast cancer, colorectalcancer, cervical cancer, gastrointestinal cancer, genitourinary cancer,head and neck cancer, lung cancer, ovarian cancer, prostate cancer,renal cancer, skin cancer, and testicular cancer. More particularly,cancers that may be treated by the compounds, compositions and methodsdescribed herein include, but are not limited to, the following: cardiaccancers, including, for example sarcoma, e.g., angiosarcoma,fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyoma;fibroma; lipoma and teratoma; lung cancers, including, for example,bronchogenic carcinoma, e.g., squamous cell, undifferentiated smallcell, undifferentiated large cell, and adenocarcinoma; alveolar andbronchiolar carcinoma; bronchial adenoma; sarcoma; lymphoma;chondromatous hamartoma; and mesothelioma; gastrointestinal cancer,including, for example, cancers of the esophagus, e.g., squamous cellcarcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma; cancers of thestomach, e.g., carcinoma, lymphoma, and leiomyosarcoma; cancers of thepancreas, e.g., ductal adenocarcinoma, insulinoma, glucagonoma,gastrinoma, carcinoid tumors, and vipoma; cancers of the small bowel,e.g., adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma; cancers of thelarge bowel, e.g., adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, and leiomyoma; genitourinary tract cancers, including, forexample, cancers of the kidney, e.g., adenocarcinoma, Wilm's tumor(nephroblastoma), lymphoma, and leukemia; cancers of the bladder andurethra, e.g., squamous cell carcinoma, transitional cell carcinoma, andadenocarcinoma; cancers of the prostate, e.g., adenocarcinoma, andsarcoma; cancer of the testis, e.g., seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, and limphoma;liver cancers, including, for example, hepatoma, e.g., hepatocellularcarcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma;hepatocellular adenoma; and hemangioma; bone cancers, including, forexample, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignantfibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignantlymphoma (reticulum cell sarcoma), multiple myeloma, malignant giantcell tumor chordoma, osteochrondroma (osteocartilaginous exostoses),benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteomaand giant cell tumors; nervous system cancers, including, for example,cancers of the skull, e.g., osteoma, hemangioma, granuloma, xanthoma,and osteitis defoinians; cancers of the meninges, e.g., meningioma,meningiosarcoma, and gliomatosis; cancers of the brain, e.g.,astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma),glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma,and congenital tumors; and cancers of the spinal cord, e.g.,neurofibroma, meningioma, glioma, and sarcoma; gynecological cancers,including, for example, cancers of the uterus, e.g., endometrialcarcinoma; cancers of the cervix, e.g., cervical carcinoma, and pretumor cervical dysplasia; cancers of the ovaries, e.g., ovariancarcinoma, including serous cystadenocarcinoma, mucinouscystadenocarcinoma, unclassified carcinoma, granulosa thecal celltumors, Sertoli Leydig cell tumors, dysgerminoma, and malignantteratoma; cancers of the vulva, e.g., squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and melanoma;cancers of the vagina, e.g., clear cell carcinoma, squamous cellcarcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and cancersof the fallopian tubes, e.g., carcinoma; hematologic cancers, including,for example, cancers of the blood, e.g., acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, andmyelodysplastic syndrome, Hodgkin's lymphoma, non Hodgkin's lymphoma(malignant lymphoma) and Waldenstrom's macroglobulinemia; skin cancers,including, for example, malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi,lipoma, angioma, dermatofibroma, keloids, psoriasis; and adrenal glandcancers, including, for example, neuroblastoma.

The compounds according to formula I or II can also be administered incombination with existing methods of treating cancers, for example bychemotherapy, irradiation, or surgery. Thus, there is further provided amethod of treating cancer comprising administering an effective amountof a compound according to formula I or II, or a salt thereof, to anindividual in need of such treatment, wherein an effective amount of atleast one further cancer chemotherapeutic agent is administered to theindividual. Examples of suitable chemotherapeutic agents include any of:abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol,altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine,bevacizumab, bexarotene, bleomycin, bortezombi, bortezomib, busulfanintravenous, busulfan oral, calusterone, capecitabine, carboplatin,carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparinsodium, dasatinib, daunorubicin, decitabine, denileukin, denileukindiftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolonepropionate, eculizumab, epirubicin, erlotinib, estramustine, etoposidephosphate, etoposide, exemestane, fentanyl citrate, filgrastim,floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib,gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelinacetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinibmesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate,lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole,lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine,methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone,nandrolone phenpropionate, nelarabine, nofetumomab, oxaliplatin,paclitaxel, pamidronate, panitumumab, pegaspargase, pegfilgrastim,pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine,quinacrine, rasburicase, rituximab, sorafenib, streptozocin, sunitinib,sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone,thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab,trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine,vincristine, vinorelbine, vorinostat, and zoledronate.

The compounds may be administered by any route, including oral, rectal,sublingual, and parenteral administration. Parenteral administrationincludes, for example, intravenous, intramuscular, intraarterial,intraperitoneal, intranasal, intravaginal, intravesical (e.g., to thebladder), intradermal, transdermal, topical or subcutaneousadministration. Also contemplated is the instillation of a drug in thebody of the patient in a controlled formulation, with systemic or localrelease of the drug to occur at a later time. For example, the drug maybe localized in a depot for controlled release to the circulation, orfor release to a local site of tumor growth. Advantageously, thecompounds are administered in the form of a pharmaceutical composition.

In another aspect, there is provided a method for predicting thesusceptibility of a cancer to treatment with PRMT5 inhibitors comprisingcontacting a cancer cell with a PRMT5 inhibitor, comparing thedistribution of PRMT5 in the cell after contacting with the distributionof PRMT5 in the cell before contacting or the distribution of PRMT5 in acontrol cell which has not been contacted with the compound to determinewhether the contacting with the compound results in an increase in therelative concentration of PRMT5 in the cytoplasm of the cell as comparedto the nucleus of the cell.

Examples High Grade Gliomas Overexpress PRMT5 and is Inversely Relatedto Survival

It has been demonstrated that epigenetic processes driven by PRMT5overexpression are relevant in regulation of key oncogenic pathways thatare operable in B-cell lymphomas and GBM. Eight patient-derived GBM celllines and 45 GBM tumors express abundant levels of PRMT5 protein.Interestingly, no low or intermediate grade astrocytomas expressed PRMT5suggesting that PRMT5 overexpression may play a pathologic roleselectively in high-grade astrocytomas. Importantly, normal brain ornormal human astrocytes did not express any measurable PRMT5. In supportof this, PRMT5 expression inversely correlated with survival of GBMpatients (FIG. 2, r=−0.57, p=0.0001) and correlated with proliferationrate of GBM cell lines (not shown, r=0.81, p<0.0001). Elevated PRMT5expression is also observed in the GBM tumors of symptomatic mice in apreclinical mouse model of GBM. Previous studies showed that PRMT5 canstimulate cell growth and proliferation and induce transformation, cellsthat overexpressed PRMT5 were able to form colonies at a rate comparableto that of MYC/RAS-transformed cells (10). These results indicate thatPRMT5 could be a potential oncogenic marker and therapeutic target forprimary and recurrent GBM.

Inhibition of PRMT5 Overexpression Leads to Apoptosis, Cell CycleArrest, and Reduced Invasiveness of GBM Cell Lines.

We developed small inhibitory RNA molecules (siRNA) that efficientlyknock-down PRMT5 expression leading to demethylation of target histoneprotein arginine residues (H4R3). SiRNA specific for PRMT5 led GBM cellsto undergo cell cycle arrest (not shown), spontaneous apoptosis andcomplete inhibition of cell migration (FIG. 3). PRMT5 silencingdisplayed a significant increase in the Bax/Bcl-2 ratio relative to theratio observed in the scramble control, validating the role of the upregulation of Bax in PRMT5 silencing induced apoptosis. Apoptosisoccurred independent of caspase enzyme activity and p53 status. PRMT5siRNA46: TGCCTATGAACTCTTTGCC; PRMT5 siRNA5: ATAGCTGACACACTAGGGG; PRMT5siRNA48: TCTCAGACATATGAAGTGT; PRMT5 siRNA-D: CCGCTATTGCACCTTGGAA.

Our results showed that PRMT5 silencing substantially reducedinvasiveness of GBM cells by transwell assay (FIG. 3B), scratch assayalso showed that migration of GBM cells significantly decreased withPRMT5 silencing. Other preliminary data showed PRMT5 to be overexpressed in the GBM-like tumors of p53/pten/Nfl haploinsufficient micethat has become an interesting preclinical model of GBM. PRMT5 knockdownled GBM cell lines to become markedly sensitized to the toxic effects oftemazolomide, an agent with antitumor activity in GBM primary tumors andin brain tumor stem cells that give rise to GBM.

Tumor Suppressor Gene ST7 and Chemokines RANTES, IP10, CXCL11 areTargeted and Silenced by PRMT5.

We used a microarray to identify potential targets of PRMT5, TSG ST7 andthree chemokines (RANTES, IP10, CXCL11) were identified to be targets ofPRMT5. Chromatin immunoprecipitation assay showed that siRNA treatmentled to loss of PRMT5 recruitment on the promoter of the ST7, RANTES,IP10, and CXCL11. FIG. 4 shows quantitative/qualitative PCR resultsusing primers that specifically amplify promoter regions of ST7 andChemokines CCL5, CXCL10 and CXCL11. Treatment of cells with siRNA toknock down PRMT5 levels shows that ChIP assays fail to amplify promoterregions indicating loss of PRMT5 recruitment on each of the examinedpromoters.

ST7, RANTES, IP10, and CXCL11 transcript and protein levels rapidlyincreased after PRA/ITS silencing FIG. 5. PRMT5 knockdown led tosecretion of the chemokines into medium, which might help to recruit NKcells and T cells to elicit immune response toward GBM tumors. The humanST7 gene was first recognized as a candidate tumor suppressor based onits chromosomal location (7q31.1) at a site of frequent loss ofheterozygosity and its reduced expression in some types of cancer. ST7induces remodeling of the extracellular matrix in other solid tumorsindicating that ST7 mediates tumor suppression by changing the tumormicroenvironment which may contribute to inhibition of invasiveness andmigration of human GBM cells by PRMT5 silencing. Collectively, our datasuggest that PRMT5 silencing may be an attractive strategy to explorealone and in combination with other agents that target epigeneticprocesses.

Comparative Modeling of Human PRMT5 and Model Validation by MolecularDocking.

siRNA technology is in the beginning stages of development. The firstphase 1 study was completed with a small micro RNA in 2009 and, whilethe technology represents a promising advance for future experimentaltherapeutic strategies, other approaches to inhibit PRMT5 activity wouldcertainly be more attractive. We therefore explored rational design ofsmall molecule compounds to inhibit PRMT5 activity. We constructed an“in silico” model since PRMT5 crystal structure has not been described.A model of human PRMT5 catalytic domain was built from availablehomologous crystal structures. The hPRMT5 sequence retrieved from NCBIsequence database (NP_(—)006100; 637 aa) was submitted as query forprotein BLAST to PDB database using NCBI blastp. Out of the severalhits, four unique templates which had approximately 40-50% similarity tothe human sequence were selected for modeling (Table 1). The templatestructures were co-crystallized with SAH, the catalytic reactionproduct. One of the crystal structures of rat PRMT1 (PDB ID: 1OR8) wasco-crystallized with a peptide arginine residue also along with SAH.

TABLE 1 BLAST result for the selected templates for human PRMT5modeling. PDB ID Protein Organism Identity Similarity Gaps Alignment2V74 CARM1 Mouse 28 44 8 329-483 2FYT PRMT3 Human 21 38 16 324-598 1F3LPRMT3 Rat 21 39 14 324-598 1OR8 PRMT1 Rat 26 48 7 359-475

The model building and refinement was carried out with MODELLER9v1 (Saliand T. L. Blundell, 1993). Structure-based sequence alignment verifiedthe highly conserved nature of this domain, especially the amino acidsclosest to the catalytic site. The residues directly participating inthe catalytic function were primarily located towards the N-terminalhalf of the catalytic domain. The automodel optimization and refinementprotocol in Modeller was used to generate 100 models with SAH in thecatalytic site. In this protocol, each model is initially optimized withthe variable target function method (VTFM) with conjugate gradients. Itis further refined using molecular dynamics with simulated annealingprotocol. Best model based on the built-in energetic criterion wasselected for structure-based virtual screening.

Structural super-positioning of the selected templates to the modelshowed that the tertiary structure is essentially conserved although thesequence identity is low. This is to be expected since all the proteinshave similar function and uses the same co-factor (SAM) for catalyticfunction. A visual inspection of the aligned catalytic site of thetemplates revealed that the key catalytic interactions are conserved inthe model as well. This include Glu392 involved in the bifurcatedhydrogen bonding with ribose hydroxyl groups of the co-factor SAM andthe Glu435 involved in hydrogen bonding with arginine of the histonesubstrate.

The final model from Modeller was taken into Schrodinger molecularmodeling environment Maestro v8.5 for preliminary preparation fordocking employing the recommended Protein Preparation Wizard protocol.Proper protonation states and hydrogen bonding network within theprotein was optimized. The structure was minimized using imperf programwith cut off criteria of 0.30A RMSD using OPLS2005 force field. Thefinal structure was carried forward for the docking experiments. A gridcube box of 25 Å size was created around the model keeping the SAH asthe center. A van der Waal's scaling of 0.9 was used in the protein gridpreparation in order to account for the soft induced fit effect. Inorder to validate the modeled active site, the non-selective PRMTinhibitor sinefungin and SAH were docked flexibly into the model usingGLIDE XP in Schrodinger suite 2008. The docking result essentiallyreproduced the crystal mode as compared by superimposing the bioactiveSAH crystal structure from its native crystal structure. (FIG. 4) TheSAH docked human PRMT model was then used to generate a grid for anotherdocking calculation to probe the close-by substrate arginine bindingsite. Docking with capped arginine residue could reproduce similarinteractions as observed in the template crystal co-ordinate (FIG. 6).These molecular docking experiments confirmed that the catalytic sitewas modeled appropriately to screen larger library of compounds to findpotential competitive enzyme inhibitors.

Virtual Screening.

The validated docking protocol was used to screen ChemBridge CNS-Set™screening library of 10,000 compounds. The compound library digitalversion in 2D SDF file format was submitted to a ligand coordinatepreparation protocol employing LigPrep program. LigPrep accounts for thedifferent tautomeric and stereoisomeric states of the compounds as wellas a low energy ring conformation sampling. Possible ionization statesof the compounds at pH 7±2 were generated using Ionizer module. Finalcoordinates were minimized by OPLS2001 molecular mechanics force fieldto obtain geometry optimized 3D coordinates. The prepared library of17,090 coordinates was screened through the HTVS (High ThroughputVirtual Screening) stage of the Virtual Screening Workflow scriptprovided with the Schrodinger suite 2008. Non-planar amide bondconformations were penalized in all the stages of the docking run. Theretained 50% of the top scored coordinates were then passed to thesecond stage of SP (Standard Precision) screening. The top 50% of thecoordinates were finally screened by the most rigorous calculation inthe XP (Extra Precision) stage. All the scored coordinates in this stagewere retained for further investigation.

Compounds with lowest binding energy from the screen were visuallyinspected for contacts that mimic conserved PRMT-SAH-ARG interactions. Aset of constraints; the requirement of ligand binding occupancy at a)the co-factor binding pocket and b) at the substrate arginine-PRMTbinding cavity; were used in the compound selection. Eight potentialcompounds were identified for biological investigation based on theirbinding energy as listed in the table above (FIG. 7) Compound 5 (bindingenergy: −8.10 kcal/mol) is shown docked into the hPRMT5 model (pinkcarbon, ball and stick representation). SAH (green carbon) and cappedarginine residue (red carbon; only side chain visible) docked bindingpositions from the model validation docking experiments are superimposedas line representation. For clarity, protein molecular surface isgenerated omitting the residues covering the catalytic site face.Hydrogen bonds to the protein are shown in yellow dotted lines.

Initial Screening of Compounds in Bioassays.

Initial Screening experiments were performed utilizing monoclonalantibodies specific for H4R3 symmetric dimethyl arginine and confocalmicroscopy generated images evaluating H4R3 methylation. Controlsincluded scrambled and SiRNA specific for PRMT5 to verify knockdown andloss of H4R3 methylation. Confocal images in FIG. 9 show that Compounds3, 5, and 8 (not shown) were capable of inhibiting H4R3 symmetric 2Mearginine methylation similar to that seen with SiRNA based inhibition ofPRMT5 expression. Initial dose titration of these compounds revealedthat these compounds also possessed similar anti proliferative activity(FIG. 7B) and promote apoptosis of GBM cell lines (not shown). Findingssimilar to PRMT5-specific SiRNA treatment. Interestingly, none of theremaining 5 compounds in our screen (FIG. 7), all of which failed tochange the symmetrical dimethylation (S2Me) of H4R3, had any toxic oranti proliferative activity on GBM cells. This observation suggestedthat the reduction in S2Me of H4R3 (and thus PRMT5 activity) correlatedwith an anticancer activity that we've observed with SiRNA specific forPRMT5.

Compound 5 appeared to demonstrate the most efficacy in blocking bothS2Mc of the PRMT5 target H4R3 and proliferation of the U1242 cell line.Therefore, we performed a series of titration experiments with compound5 to evaluate if a dose response was observed at both degree of reducedS2Me and proliferation rate. Following addition of DMSO media control orcompound 5 (20 uM, 30 uM, 40 uM, 50 uM and 100 uM). FIG. 10 showsabsolute cell numbers determined at 48 hours after addition of eithercontrol or various concentrations of compound 5 identified in ourinitial screen. Interestingly, a dose response was seen at both cellularproliferation and the degree of H4R3 methylation. Compound 5 wastherefore chosen as our lead compound around which we would direct ourfocus on verifying selectivity of PRMT5 enzyme inhibition as well asfuture strategies to enhance its potency and activity against high gradeglioma tumors. Preliminary enzymatic activity assays are currentlyunderway and will be provided as an update during submission ofsupplementary materials.

After determining that compound 5 demonstrated the best activity (byinhibition of S2M3-H4R3), we next wished to explore whether compound 5was capable of selectively inhibiting a type II PRMT enzyme. Do achievethis, we performed enzyme inhibition assays using purified PRMT1 (a typeI PRMT enzyme) and purified PRMT5 (a type II PRMT enzyme). We were ableto demonstrate in 3 replicate experiments, that compound 5 was capableof selectively inhibiting PRMT5 activity (and not PRMT1 activity FIG.11, p<0.0001).

Inhibiting PRMT5 with Compound 5 is Improved with HDAC Inhibition.

Recent work reported by Pal et al has shown that PRMT5 associates withSWI/SNF chromatin remodeling complexes along with other co-repressormolecules like HDAC2. Biochemical assays have demonstrated that PRMT5activity on target H4R3 and H3R8 histone arginine residues is markedlyenhanced when lysine residues become deacetylated by HDAC enzymes. Wetherefore wished to sec if we could achieve improved PRMT5-inhibitoryactivity by co-treating GBM cell lines with low doses of HDAC inhibitors(TSA, 75, 100 nM) that have been shown to result in acetylation oflysine residues neighboring PRMT5 target arginine residues. FIG. 12shows the results of these studies. We were able to achieve loss of S2Mestatus of H4R3 at much lower (>10-fold) concentrations of compound 5 (10uM) when cells were co-treated with the HDACi TSA. We utilized a flowcytometric assay to evaluate symmetric dimethyl H4R3 (S2Me-H4R3) contentand confocal microscopy to verify loss of methylation status. Anti tumoractivity is also enhanced when PRMT5 inhibitors are used in combinationwith HDAC inhibitor drugs.

Generating More Potent PRMT5 Inhibitors.

To generate more potent and selective small molecule compounds toinhibit PRMT5 activity. The ability to achieve equivalent or improvedenzyme inhibition at much lower concentrations will likely enhancespecificity of our reagent while minimizing the likelihood of toxicityduring preclinical evaluation in our in vitro and in vivo developmentplatforms.

These experiments utilized compound 5 as the “backbone compound” andmodification of R groups to allow for various permutations that willenhance inhibitory activity and potency (FIG. 13). Consequently, theseexperiments initially produced additional candidate compounds forevaluation. It may be necessary to further optimize compounds to allowfor improved bio availability, CNS blood brain barrier penetration, orreduced toxicity. A combination of structure based computational effortsand synthetic medicinal chemistry approaches will be used to continueour search for an optimal compound. The computationally selectedcandidates will be synthesized and evaluated for biological activity.Compound 5 is commercially available; however, the synthetic scheme ofthe compound has never been reported. We synthesized compound 5 througha one step reductive amination reaction. All the optimized analogs canbe synthesized with the similar procedure as compound 5.

Computational Optimization of Lead Compound 5.

Two similar approaches will be used for the structure based leadcompound optimization; 1) AlleGrow uses a grow algorithm on the bindingsite utilizing a pre-assigned chemical fragment library, 2) CombiGlideuses commercially available synthetic reagent library. The bindingfeatures of the lead compound 5 (ChemBridge ID 9033823) shows keyfavorable van der Waal's, aromatic, cation-pi and H-bonding interactionsto the hPRMT5 catalytic site (FIG. 13).

Preliminary combinatorial optimization of the lead compound 5 usingAlleGrow already identified a few novel derivatives with Glide XPscoring energies about 1-3 kcal/mol stronger in binding than the leadcompound itself. Keeping R2 fixed, several ring structures weresubstituted for R1 and their binding affinity were evaluated using GlideXP. Similarly, R1 was fixed and R1 substitution chemical space wasexplored. Out of the several tens of thousands of combinationsevaluated, 7 potential compounds with improved binding energies havebeen determined and are identified for chemical synthesis. (FIG. 14).Additional compounds with equivalent binding energies have been designedand synthesized and are presently under evaluation.

A larger and diverse synthetic chemical space are presently beingcomputationally explored by utilizing commercially available chemicallibrary. Schrodinger CombiGlide program will be used for this approach.A comprehensive database of commercially available compounds is providedfree for academic use by UCSF (ZINC; zinc.docking.org). Current versionof ZINC (v 8.0) database contains more than 8 million compounds. Thefragment like subset of ZINC currently stores 453,539 compounds. Thereagent files prepared from these compound fragments will becombinatorially attached to R1 and R2 substitution positionsrespectively to generate several hundreds of thousands of compounds.These virtual compounds are docked to the catalytic site to evaluate thebinding energy (FIG. 15). Several compounds with better energy comparedto the original compound 5 have been selected for chemical synthesis andbiological evaluation FIG. 14). Combinatorially optimized novel R2substituted compounds in the binding pocket (FIG. 15 b) as compared tothe binding of compound 5, SAH and substrate arginine residue (FIG. 15a). R2 Optimized compounds with enhanced binding affinity showadditional hydrogen bonding to Glu444 carboxylate oxygen and Leu437backbone carbonyl oxygen.

Characterization of PRMT5 Enzyme Inhibition with Lead Compound 5 (CMP5).

The screening of our first candidate compounds identified by our PRMT5in silico model showed CMP5 to have the most profound effects asdemonstrated by loss of symmetric dimethyl-H4R3 and antiproliferativeeffect in astrocytoma cells (FIGS. 7 & 8). The next step requiredstudies to explore whether CMP5 was capable of selectively inhibiting atype II PRMT enzyme. Type I PRMT enzymes are involved in transcriptionalactivation (asymmetric dimethylation at H4R3) and type II PRMT enzymesregulates transcriptional repression (symmetric dimethylation or S2Mc atH4R3). To achieve this goal, we performed enzyme inhibition assays usingpurified PRMT1 (type I PRMT enz) and purified PRMT5 (type II PRMT enz).Assays were performed utilizing 2 methods. The first utilized methodsdescribed previously by Zhao et at (Nat Struct Mol Biol. 2009), where 1ug of H4 (Roche) was used as substrate and 2 uCi ofS-adenosyl-L-methyl-³H-methionine (³H-SAM; Amersham) as the methyl donorin the presence or absence of 100-500 uM of CMP5 was incubated in HMTasebuffer for 3 h at 37° C. Reaction mixtures were spotted on Whatman P-81filter paper and washed to remove unincorporated [³H]SAM. Methylatedpeptides were detected by scintillation counting. This method showedsignificant inhibition of methyltransferase activity (55%reduction±7.8%, p<0.001, data not shown). The second method utilized amethyltransferase activity assay to distinguish if differentialinhibitory activity of CMP5 existed against the type I PRMT1 vs. thetype II enzyme PRMT. The methyltransferase assay we used (Amsbio inc)utilized purified PRMT5 and PRMT1 enzyme preparations and tested formethylation of Histone H4 at arginine 3 (H4R3). SAM was incubated with asample containing substrate H4 peptide, different concentrations ofmethyltransferase enzymes (PRMT5 or PRMT1), in presence or absence ofCMP5 (100-500 uM) for one hour at 37° C. Fluorescently labeled SAMtracer was added followed by anti-SAM antibody that produces a change influorescent polarization that can be measured using a fluorescencereader. We were able to demonstrate in 3 replicate experiments, thatCMP5 was capable of selectively inhibiting PRMT5 activity and not PRMT1enzymatic activity (FIG. 16, p<0.0001). Because of the differentialactivities of these two distinct classes of PRMT enzymes, the outcome ofour inhibitor experiments has important implications. We know that PRMT5is selectively over expressed in high grade astrocytomas (FIG. 2), worksto silence both tumor suppressor genes and proinflammatory cytokines,and is directly involved with promoting malignant cell growth andsurvival. Discovery of a small molecule inhibitor with selective type IIPRMT enzyme inhibitory activity at low micro molar range, has allowed usto now direct our focus on improving the potency and selectivity of ourlead compound (CMP5). It has also allowed us to pursue studiesinvestigating anti tumor activity of this compound (below).

Design and Synthesis of a Second Generation of CMP5 Analogs (BLL2-BLL8).

Specific Aim 1 of our proposal outlines a strategy to design andsynthesize compounds with more potent and selective PRMT5 inhibitoryactivity. We have designed and synthesized 7 additional analogs of CMP5(BLL2-BLL8). The design of the analogs is based on replacing thepyridine ring of CMP5 with different substituted benzene. BLL-2-BLL-8were synthesized through a one step reductive amination reaction (FIG.17) similar to the synthesis of CMP5. Presently we have synthesized mgquantities of CMP5 (BLL1) and BLL 2-8 and are now screening forselective PRMT5 (vs PRMT1) inhibitory activity in our bioassays andmethyltransferase enzyme assays.

PRMT5 Inhibition and Anti Tumor Activity is Enhanced when Used inCombination with Histone Deacetylase (HDAC) Inhibitors.

The biochemical association of PRMT5 and HDAC2 with repressive chromatinremodeling complexes and cooperative enzyme kinetics has been previouslydescribed. FIG. 9 shows that S2Mc-H4R3 is unchanged in astrocytoma celllines treated with single agent HDAC inhibitor (TSA) or CMP5 (PRMT5inhibitor at 25 uM), however, when drugs are combined, the reduction ofS2Me at H4R3 occurred in a synergistic fashion. Importantly, theseresults were achieved at concentrations of CMP5 that are log fold lowercompared to our original dose response experiments where 100 uM CMP5 wasrequired for reduced methylation (see FIG. 8). To evaluate theconsequences of this enhanced loss of S2Me-H4R3 on histone 4, weperformed an evaluation of cellular apoptosis at 24, 48 and 72 hoursafter plating astrocytoma cell lines (U1242 and U251) in presence ofsimilar concentrations of TSA (25 nM) and/or CMP5 (25 uM). As a control,we utilized a flow cytometric assay to evaluate symmetric dimethyl H4R3(S2Me-H4R3, FIG. 18) content and confocal microscopy (not shown) toverify decrease of methylation status. We used Aimexin V/PI staining andflow cytometry to examine apoptosis (below). In FIG. 18, use of singleagent CMP5 (green) or TSA (blue) showed no change in S2Me-H4R3, howevercombination treatment (red) showed significant loss of methylation. Weare presently performing dose response experiments to examine whetherthis activity can be improved. In data not shown, we can achieve similarresults with lower doses of CMP5 (10 uM). Anti tumor, proapoptoticactivity was enhanced in a synergistic fashion when low dose CMP5 wasused in combination with the HDAC inhibitor TSA (FIG. 19). InhibitionPRMT5 with CMP5 and HDAC enzymes (at 10 fold lower concentrations) leadsto synergistic induction of apoptosis of astrocytoma cell lines.

Synergistic Effect of the Combination of CMP5, Trichostatin A (HDACInhibitor) and 5-Azacytidine (DNA Methyltransferase Inhibitor) inPromoting Cell Death.

Because PRMT5 associates with other transcriptional co-repressors suchas HDAC2 and DNMT3a, evaluation of synergistic effects between HDAC andDNA methylation inhibitors and PRMT5 inhibitors was performed in vitro.We have observed that sub toxic doses of DNA methyltransferase inhibitor5-aza, HDAC inhibitor Trichostatin A (TSA), and PRMT5 inhibitor CMP5 ledto synergistic induction of cell death (by flow cytometry, FIG. 20A) andloss of S2Me-H4R3 (by confocal microscopy and western blot) (FIGS. 20Band 20C).

Development of More Potent and Selective Analogs of CMP5.

The lead compound identified in our initial screen showed an IC50 inmicromolar (uM) ranges. Thus, a structure-based computationalcombinational lead optimization (FIG. 22) was used to design more potentanalogs. Forty CMP5 analogs were designed and synthesized, one of theanalogs (BLL36) was shown to be more selective and potent than CMP5 interms of ability to inhibit methyltransferase activity (FIG. 21A) and toinduce cell death (FIG. 21B).

1. A compound of formula (I)

wherein R₁ is

R₂ is

A₁, A₂, A₃, and A₄ are each individually hydrogen, halo, alkyl, alkoxyl,—OH, —NH₂, or —NO₂; A₈, A₉, A₁₀, A₁₁, A₁₂, A₁₃, and A₁₄ are eachindividual hydrogen and A₁₅ is C₁-C₆ alkyl; or a salt thereof. 2-4.(canceled)
 5. A pharmaceutical composition comprising a therapeuticallyeffective amount of the compound of claim 1, in combination with apharmaceutically suitable carrier.
 6. The pharmaceutical compositionaccording to claim 5 further comprising at least one histone deacetylase(HDAC) inhibitor.
 7. The pharmaceutical composition according to claim 5further comprising at least one hypomethylating agent.
 8. A method ofinhibiting protein arginine methyltransferase 5 (PRMT5), comprisingcontacting the compound of claim 1 with protein argininemethyltransferase 5 (PRMT5), thereby inhibiting protein argininemethyltransferase 5 (PRMT5).
 9. A method of treating cancercharacterized by an overexpression of protein arginine methyltransferase5 (PRMT5) in a mammal, comprising administering to the mammal, atherapeutically effective amount of the compound of claim
 1. 10.(canceled)
 11. The compound according to claim 1, wherein A₁, A₂, A₃,and A₄ are each individually hydrogen, halo, methyl, or methoxy.
 12. Thecompound according to claim 1, wherein the compound is selected from thegroup consisting of:


13. A pharmaceutical composition comprising the compound of claim 12.14. The pharmaceutical composition of claim 13 further comprising atleast one histone deacetylase (HDAC) inhibitor.
 15. The pharmaceuticalcomposition according to claim 13 further comprising at least onehypomethylating agent.
 16. The method according to claim 8, wherein thecompound is:


17. The method according to claim 8, further comprising determining theactivity of the protein arginine methyltransferase 5 (PRMT5).
 18. Themethod according to claim 17, wherein the protein argininemethyltransferase 5 (PRMT5) is a purified enzyme preparation.
 19. Themethod according to claim 17, wherein the protein argininemethyltransferase 5 (PRMT5) is a cell.
 20. The method according to claim19, wherein the activity is determined in the presence of an HDACinhibitor.
 21. The method according to claim 9, wherein the cancer is anastrocytoma.
 22. The method according to claim 9, wherein A₁, A₂, A₃,and A₄ are each individually hydrogen, halo, methyl, or methoxy.
 23. Themethod according to claim 9, wherein the compound is:


24. A method of treating cancer characterized by an overexpression ofprotein arginine methyltransferase 5 (PRMT5) in a mammal, comprisingadministering to the mammal, a therapeutically effective amount of thepharmaceutical composition of claim
 5. 25. A method of treating cancercharacterized by an overexpression of protein arginine methyltransferase5 (PRMT5) in a mammal, comprising administering to the mammal, atherapeutically effective amount of the pharmaceutical composition ofclaim
 6. 26. A method of treating cancer characterized by anoverexpression of protein arginine methyltransferase 5 (PRMT5) in amammal, comprising administering to the mammal, a therapeuticallyeffective amount of the pharmaceutical composition of claim 7.